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Laser Capture Microdissection of muscle fiber populations and expression analysis by RT-PCR

◊ Horizontal gel chamber
◊ Power supply
◊ Gel Doc 2000TM Documentation System (Bio-Rad, Cat. # 170-8615)


RNase-free Technique
In addition to the usual precautions listed below, some special precautions were taken connected to the rapid staining protocol:
1. Use RNase AWAY according to the manufacturers instructions on laboratory bench surfaces, cryostat, cryostat knife, PixCell II Laser Capture Microdissection System and Arcturus alignment tray.
2. Disposable gloves are to be frequently changed and RNase-free plasticware used.
3. Heat inactivate serum.
4. Use DEPC-treated water for the preparation of all staining solutions and for washing.
5. Use chemicals in the highest grade available.
6. Staining and washing time is to be reduced to a minimum, while still preserving the capability to distinguish different muscle fiber populations.


Method
1. After dissection, limb muscle and EOMs are covered in OCT compound and successively frozen in cooled 2methylbutane (30 seconds) and liquid nitrogen (10 minutes). The muscle can be stored at -80C.

2. The muscle is cut into 10 m sections with the cryostat. During the cutting process, the slides with 4 sections each are stored on dry ice and are afterwards stored at -80C.

3. For the acetylcholine esterase stain by Karnowsky and Roots, the following solution is prepared in the following order:
5 mg of acetylthiocholine iodide are dissolved in 6.5 ml of 0.1 M sodium acetate buffer pH 5.2, followed by:
0.5 ml 0.2 M sodium citrate,
1.0 ml of 30 mM cupric sulfate,
1.0 ml DEPC-treated water, and
1.0 ml of 5 mM potassium ferricyanide.

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